Solvent-assisted trypsin digestion of ricin for forensic identification by LC-ESI MS/MS

The castor bean plant (Ricinus communis) is used in large quantities for oil production and is also a common ornamental garden plant. However, the beans contain 1-3% of the highly toxic protein ricin, a type II ribosome-inactivating protein that is covered by the Chemical Weapons Convention, and there have been a number of reports concerning the use, or alleged use, of the toxin in terrorist and criminal activities. In the study reported here, we investigated the potential utility of organic solvent-assisted trypsin digestion of crude extracts containing the closely related toxins ricin or abrin to prepare samples for peptide analysis by liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Diagnostic tryptic fragments of the toxins were detected and unambiguously identified by this procedure. The sample preparation protocol substantially reduces the sample preparation time, from overnight to an hour, and thus greatly reduces the total time required for analyses, to less than 2 h. Furthermore, the reported procedure leaves the disulfide bonds in the protein intact. This is highly relevant in the context of the Chemical Weapons Convention, since the disulfide bond connecting the two chains of ricin indicates the presence of an intact toxin and provides additional forensic evidence for the analytical results.